Gene Editing for Duchenne Muscular Dystrophy Using the CRISPR/Cas9 Technology: The Importance of Fine-tuning the Approach

To the editor: Four articles have been published recently that describe the use of CRISPR/Cas9 technology to mutate the Duchenne muscular dystrophy (DMD) gene.1Nelson CE Hakim CH Ousterout DG Thakore PI Moreb EA Rivera RM et al.In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.Science. 2016; 351: 403-407Crossref PubMed Scopus (805) Google Scholar,2Tabebordbar M Zhu K Cheng JK Chew WL Widrick JJ Yan WX et al.In vivo gene editing in dystrophic mouse muscle and muscle stem cells.Science. 2016; 351: 407-411Crossref PubMed Scopus (740) Google Scholar,3Long C Amoasii L Mireault AA McAnally JR Li H Sanchez-Ortiz E et al.Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.Science. 2016; 351: 400-403Crossref PubMed Scopus (671) Google Scholar,4Xu L Park KH Zhao L Xu J El Refaey M Gao Y et al.CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice.Mol Ther. 2016; 24: 564-569Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar In each study the authors deleted exon 23 of the mdx mouse by inducing double-strand breaks in the introns flanking this exon. Exon 23 contains a multiple of three nucleotides and a nonsense mutation. Thus, removal of exon 23 maintains the normal reading frame and restores dystrophin (Dys) expression as the nonsense mutation is deleted. However, the resulting Dys protein does not possess proper structure because of disruption of the spectrin-like repeats (SLRs) within the central rod domain. The amino acid junctions of the α-helices do not correlate with the DNA sequence of the exons, and when exon 23 is deleted there is a fusion of helices A and B of repeat 6 with the helix A of repeat 7, thereby missing a helix C. These studies represent proof of principle that CRISPR/Cas9 technology can be used to correct the DMD gene and to restore the expression of an internally truncated Dys protein. However, this exon-deletion strategy will in most cases not result in an adequate SLR structure, as has been the case for the exon-skipping strategy using antisense oligonucleotides (AONs), which failed during clinical trials. The 2.4-Mb DMD gene contains 79 exons coding for a very complex Dys protein.5van Westering TL Betts CA Wood MJ Current understanding of molecular pathology and treatment of cardiomyopathy in Duchenne muscular dystrophy.Molecules. 2015; 20: 8823-8855Crossref PubMed Scopus (69) Google Scholar The central part of the protein is made of a rod domain containing 24 SLRs, each comprising three α-helixes (A, B, and C) forming a coiled-coil structure.6Nicolas A Raguenes-Nicol C Ben Yaou R Ameziane-Le Hir S Cheron A Vie V et al.Becker muscular dystrophy severity is linked to the structure of dystrophin.Hum Mol Genet. 2015; 24: 1267-1279Crossref PubMed Scopus (49) Google Scholar,7Ameziane-Le Hir S Paboeuf G Tascon C Hubert JF Le Rumeur E Vie V et al.Dystrophin hot-spot mutants leading to Becker muscular dystrophy insert more deeply into membrane models than the native protein.Biochemistry. 2016; 55: 4018-4026Crossref PubMed Scopus (2) Google Scholar Each α-helix has a heptad structure with hydrophobic amino acids located in positions 1 and 4. The limits of the coding sequences of these helices do not correspond precisely to the limits of the exons. Whereas the N-terminal region of Dys is required for interacting with actin, a portion of exons 42–45 is required to interact with nNOS, and the C-terminal region interacts with the dystroglycan complex and syntrophin.8Lai Y Zhao J Yue Y Duan D a2 and a3 helices of dystrophin R16 and R17 frame a microdomain in the a1 helix of dystrophin R17 for neuronal NOS binding.Proc Natl Acad Sci USA. 2013; 110: 525-530Crossref PubMed Scopus (68) Google Scholar Seventy percent of DMD patients have a deletion of one or more exons within the DMD gene that leads to a premature stop codon and the absence of the Dys protein.9Bladen CL Salgado D Monges S Foncuberta ME Kekou K Kosma K et al.The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations.Hum Mutat. 2015; 36: 395-402Crossref PubMed Scopus (388) Google Scholar Patients with Becker muscular dystrophy (BMD) also have a deletion of one or several exons in the DMD gene,7Ameziane-Le Hir S Paboeuf G Tascon C Hubert JF Le Rumeur E Vie V et al.Dystrophin hot-spot mutants leading to Becker muscular dystrophy insert more deeply into membrane models than the native protein.Biochemistry. 2016; 55: 4018-4026Crossref PubMed Scopus (2) Google Scholar but because the latter deletions do not cause a frameshift, an internally deleted Dys protein is translated. These BMD patients nevertheless have more or less severe symptoms, depending on the structure of the resulting Dys.6Nicolas A Raguenes-Nicol C Ben Yaou R Ameziane-Le Hir S Cheron A Vie V et al.Becker muscular dystrophy severity is linked to the structure of dystrophin.Hum Mol Genet. 2015; 24: 1267-1279Crossref PubMed Scopus (49) Google Scholar As demonstrated by Le Rumeur's group, the expression of a Dys protein with an inadequate SLR leads to a severe BMD,6Nicolas A Raguenes-Nicol C Ben Yaou R Ameziane-Le Hir S Cheron A Vie V et al.Becker muscular dystrophy severity is linked to the structure of dystrophin.Hum Mol Genet. 2015; 24: 1267-1279Crossref PubMed Scopus (49) Google Scholar especially when the interaction with nNOS is abrogated.5van Westering TL Betts CA Wood MJ Current understanding of molecular pathology and treatment of cardiomyopathy in Duchenne muscular dystrophy.Molecules. 2015; 20: 8823-8855Crossref PubMed Scopus (69) Google Scholar In the mdx mouse model only SLR 6 is affected by exon 23 removal, whereas most of the mutations in DMD patients are located in the hot-spot region containing exons 45–55 corresponding to SLRs 16–22. nNOS is known to interact with SLRs 16 and 17, and its correct interaction with Dys seems to prevail in BMD patients with less severe phenotypes. A potential treatment for DMD involves therapeutic exon skipping. AONs are used to remove the exon flanking the patient deletion during splicing of the messenger RNA, so as to restore the normal reading frame and thus allow the expression of an internally deleted Dys.10Aartsma-Rus A Overview on DMD exon skipping.Methods Mol Biol. 2012; 867: 97-116Crossref PubMed Scopus (43) Google Scholar This approach has two drawbacks: (i) AONs must be readministered throughout the patient's life, and (ii) as for BMD, this approach may lead to a Dys protein containing an abnormal SLR and therefore a dysfunctional protein. Despite more than 360 articles on this approach in mice and cultured cells, a phase III clinical trial was halted by GlaxoSmithKline, because it failed to significantly improve the patient phenotype.11Merlini L Sabatelli P Improving clinical trial design for Duchenne muscular dystrophy.BMC Neurol. 2015; 15: 153Crossref PubMed Scopus (23) Google Scholar The failure of this clinical approach may be due in part to the fact that exon skipping aims to restore the reading frame of the dystrophin messenger RNA without paying attention to the structure of the resulting SLRs of the Dys protein. Alternative hypotheses are insufficient AON dosing or efficiency, potential toxicity of the AONs, poor delivery to the heart, and difficulties in reversing preexisting muscle weakness. However, it should be noted that the commercialization of an AON treatment was recently approved by the US Food and Drug Administration following a phase I clinical trial on only 12 patients. This approval was quite controversial.12Ledford H US government approves controversial drug for muscular dystrophy.Nature News. 20 September 2016; 15http://www.nature.com/news/us-government-approves-controversial-drug-for-muscular-dystrophy-1.20645Date: 2016Google Scholar We recently published a fine-tuned approach using the CRIPSR/Cas9 technology to correct the DMD gene.13Iyombe-Engembe JP Ouellet DL Rousseau J Chapdelaine P Tremblay JP Efficient restoration of the dystrophin gene reading frame and protein structure in DMD myoblasts using the CinDel method.Mol Ther Nucleic Acids. 2016; 5: e283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Instead of cutting within introns, we induced double-strand breaks in the exons that flank the patient deletion. Careful selection of the target sequences for the guide RNAs made it possible to delete the intron sequence and parts of these exons, resulting in the formation of a hybrid exon that not only restores the normal reading frame but also encodes a Dys protein with an adequate SLR containing a normal succession of helices A, B, and C—and thus a correct heptad structure—and producing a protein that functions adequately. Figure 1 illustrates such a hybrid SLR formed by cutting in exons 50 and 54 with the CRISPR/Cas9 technique.13Iyombe-Engembe JP Ouellet DL Rousseau J Chapdelaine P Tremblay JP Efficient restoration of the dystrophin gene reading frame and protein structure in DMD myoblasts using the CinDel method.Mol Ther Nucleic Acids. 2016; 5: e283Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar CRISPR/Cas9 technology may lead to the development of a permanent treatment for DMD. Future experiments developing CRISPR/Cas9 therapy for DMD will have to be done in an adequate mouse model such as the hDMD/mdx mouse,14't Hoen PA de Meijer EJ Boer JM Vossen RH Turk R Maatman RG et al.Generation and characterization of transgenic mice with the full-length human DMD gene.J Biol Chem. 2008; 283: 5899-5907Crossref PubMed Scopus (58) Google Scholar containing in the mdx background the human DMD gene with deletion of one or several exons as observed in DMD patients. It will be essential to demonstrate that, following systemic delivery of the Cas9 gene and two guide RNAs with an adeno-associated viral vector, there is correction of a sufficient percentage of DMD genes to rescue the dystrophic phenotype. Moreover, as indicated by VandenDriessche and Chuah,15VandenDriessche T Chuah MK CRISPR/Cas9 flexes its muscles: in vivo somatic gene editing for muscular dystrophy.Mol Ther. 2016; 24: 414-416Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar the Cas9 protein has to be expressed only transiently to avoid an immune response and accumulation of off-target mutations.

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